Clostridia are gram-positive, spore forming anaerobic bacteria. Pathogenic Clostridia species produce protein toxins of which the group of large clostridial cytotoxins (LCTs) consists of very large toxins with high in vivo toxicity as well as high structural and sequence homology (von Eichel-Streiber et al., 1996). C. difficile toxins A and B are the major cause of C. difficile pathogenicity. For a long time toxin A was considered the major virulence factor, but increasing amount of evidence is showing that in fact toxin B plays a major role in C. difficile infections (Lyras et al., 2009; Carter et al., 2012).
C. difficile toxin A and B are encoded by genes tcdA and tcdB, respectively, and the genes are located in a ˜19.6 kb pathogenicity locus (PaLoc). The PaLoc contains also two regulatory genes, namely tcdC and tcdR, which act as negative and positive regulators, respectively, of toxin expression. tcdE, also included in the PaLoc, encodes for a holin-like protein necessary for toxin A and B secretion. In non-toxigenic strains the PaLoc is replaced by a 115 bp sequence (Braun et al., 1996). DNA amplification has been used for detection of toxigenic C. difficile strains (Wren et al., 1990; McMillin et al., 1991; McMillin et al., 1992).
An isothermal DNA amplification process relying on an upstream primer, a downstream primer, and a strand invasion system is described in WO 2009/150467.